Plant Cell Advance Publication. Published on May 20, 2016, doi:10.1105/tpc.16.00011
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The starch granule-associated protein EARLY STARVATION1 (ESV1) is required
2
for the control of starch degradation in Arabidopsis thaliana leaves
3 4
Doreen Feikea1, David Seungb1, Alexander Grafa2, Sylvain Bischofb3, Tamaryn Ellickb,
5
Mario Coirob, Sebastian Soykb4, Simona Eickeb, Tabea Mettler-Altmannb5, Kuan Jen
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Lub, Martin Tricka, Samuel C. Zeemanb, Alison M. Smitha6
7 8 9 10
a
John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom
b
Department of Biology, Eidgenössische Technische Hochschule Zürich,
Universitätstrasse 2, CH-8092 Zürich, Switzerland
11 12 13 14 15 16 17 18 19 20 21
1
These authors contributed equally to the work
2
Current address: Department of Molecular Plant Physiology, Max Planck Institute for
Molecular Plant Physiology, D-14476 Potsdam, Germany 3
Current address: Department of Molecular, Cell and Developmental Biology, University
of California at Los Angeles, Los Angeles, CA 90095, USA 4
Current address: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring
Harbor, NY 11724, USA 5
Current address: Institute of Plant Biochemistry, Cluster of Excellence on Plant
Sciences (CEPLAS), Heinrich-Heine-University, 40225 Düsseldorf, Germany 6
Address correspondence to
[email protected].
22 23 24 25
Short title: Control of leaf starch breakdown
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The author responsible for distribution of materials integral to the findings presented in
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this article in accordance with the policy described in the Instructions for Authors
30
(www.plantcell.org) is: Alison Smith (
[email protected]).
31 1 ©2016 American Society of Plant Biologists. All Rights Reserved.
32
SYNOPSIS
33
Two proteins present in leaf starch granules are important for the control of starch
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turnover, allowing plants to match the depletion of starch reserves to the length of the
35
night.
36 37
ABSTRACT
38 39
To uncover components of the mechanism that adjusts the rate of leaf starch
40
degradation to the length of the night, we devised a screen for mutant Arabidopsis
41
thaliana plants in which starch reserves are prematurely exhausted. The mutation in
42
one such mutant, named early starvation 1 or esv1, eliminates a previously-
43
uncharacterized protein. Starch in mutant leaves is degraded rapidly and in a non-linear
44
fashion, so that reserves are exhausted 2 h prior to dawn. The ESV1 protein and a
45
similar uncharacterized A. thaliana protein (named Like ESV1, or LESV) are located in
46
the chloroplast stroma and also bound into starch granules. The region of highest
47
similarity between the two proteins contains a series of near-repeated motifs rich in
48
tryptophan. Both proteins are conserved throughout starch-synthesizing organisms,
49
from angiosperms and monocots to green algae. Analysis of transgenic plants lacking
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or over-expressing ESV1 or LESV, and of double mutants lacking ESV1 and another
51
protein necessary for starch degradation, leads us to propose that these proteins
52
function in the organization of the starch granule matrix. We argue that their
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misexpression affects starch degradation indirectly, by altering matrix organization and
54
thus accessibility of starch polymers to starch-degrading enzymes.
55 56
INTRODUCTION
57 58
Normal growth rates in many plants are dependent upon the accumulation of starch as
59
a product of photosynthesis during the day and its controlled utilisation as a carbon
60
source for growth during the night. In Arabidopsis thaliana leaves, up to half of the
61
photosynthetically assimilated carbon accumulates as starch in leaf chloroplasts. At 2
62
night, conversion of starch to sucrose proceeds at a near linear rate, such that about
63
95% of starch is consumed by dawn (Gibon et al., 2004; Gibon et al., 2009; Graf et al.,
64
2010; Sulpice et al., 2014). This pattern of starch biosynthesis and consumption is
65
adjusted in response to changing day length in a manner that optimises the allocation of
66
carbon for growth across a wide range of carbon availability. As day length decreases,
67
more photosynthate is allocated to starch in the day, and its rate of consumption at night
68
decreases so that supplies always last until dawn (e.g. Chatterton and Silvius, 1981;
69
Gibon et al., 2009; Sulpice et al., 2014). Adjustments are also made in response to
70
abrupt, unexpected alterations in day length (Lu et al., 2005). When plants grown in 12
71
h light – 12 h dark cycles are subjected to darkness after only 8 h of light, starch
72
degradation is slower than in a normal night so that reserves again last almost precisely
73
until dawn (Graf et al., 2010). Similarly, when the onset of darkness is delayed by 4 h,
74
starch degradation is faster so that reserves are again exhausted almost precisely at
75
dawn (Scialdone et al., 2013). Variation in the amount of starch present at the end of
76
the day also leads to adjustment of starch degradation rates to permit exhaustion of
77
reserves almost precisely at dawn (Graf et al., 2010, Scialdone et al., 2013).
78 79
We previously determined through experimental and chemical kinetic modelling
80
approaches that the adjustment of starch degradation to match the length of the night is
81
dependent on both the circadian clock and leaf starch content (Graf et al., 2010;
82
Scialdone et al., 2013). It is not known exactly how leaf starch content is perceived such
83
that it influences degradation rate. However, several potentially important features of
84
leaf starch granules are known to be under tight control. These include the timing of
85
granule initiation during leaf development, granule size, shape and number per
86
chloroplast (and therefore surface area), and the degree of crystallinity of the
87
constituent glucan polymers (Roldán et al., 2007; Szydlowski et al., 2009; Crumpton-
88
Taylor et al., 2012; Crumpton-Taylor et al., 2013; Pfister et al., 2014). These features
89
may combine with more specific control mechanisms that modulate the activities of the
90
enzymes of starch breakdown to confer the observed adjustment of the degradation
91
rate to match the length of the night.
3
92
To discover new components that influence starch degradation and mediate adjustment
93
of the rate according to the length of the night, we devised a forward genetic screen to
94
identify mutants defective in this adjustment. The screen made use of the observation
95
that if starch reserves are exhausted prior to dawn, plants rapidly exhibit large
96
transcriptional changes indicative of starvation. This phenomenon is seen in wild-type
97
plants when subjected to an extended night, beyond the point at which starch is
98
exhausted (Gibon et al., 2006), and in the short-period circadian clock mutant cca1 lhy
99
(circadian clock associated1 late elongated hypocotyl), which exhausts its starch
100
reserves about 3 h before dawn when grown in 12 h light – 12 h dark cycles (Graf et al.,
101
2010). Mutant plants unable to synthesize or to degrade starch have a transcriptional
102
signature of starvation throughout most of the night. We previously generated a
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starvation reporter line of Arabidopsis harbouring a luciferase gene fused to the
104
promoter of a gene that is expressed only after starch reserves are exhausted.
105
Luciferase expression was very low during the normal day-night cycle but increased
106
rapidly after a 2-h extension of the night (Graf et al., 2010). We reasoned that
107
mutagenesis of the reporter line followed by screening for plants with abnormal
108
temporal patterns of luciferase expression should identify mutants defective in the diel
109
control of carbon availability, including aspects of starch granule biosynthesis and
110
degradation, elements of the circadian clock necessary for correct anticipation of dawn,
111
and components that sense or signal carbohydrate status. In the current study, we
112
validated this approach by showing that, as anticipated, the screen identified previously-
113
known classes of mutants defective in starch accumulation or with reduced starch
114
crystallinity.
115 116
The screen also identified a mutant with inappropriately rapid starch degradation at
117
night, such that starch was exhausted well before dawn. We named the mutant early
118
starvation1 (esv1-1). We established that the causal mutation was in an unannotated
119
gene, encoding a protein with no known or predicted role. The protein is highly
120
conserved and tryptophan-rich and is present in the chloroplast stroma and within
121
starch granules. Based on phenotypic characterization of mutant and over-expressing
122
lines, we propose that the protein has an unrecognized and central function in the 4
123
organization of glucan polymers within starch granules. We speculate that abnormal
124
levels of the protein alter the starch granule matrix such that the mechanisms controlling
125
the rate of starch degradation no longer function correctly. We show that another,
126
related protein is also likely to be involved in organization of glucan polymers within the
127
granule matrix and can indirectly influence the control of degradation. These results are
128
particularly surprising because it has been widely assumed that the formation of the
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starch granule matrix is a physical process involving self-organization of glucan
130
polymers (Waigh et al., 1998; Waigh et al., 2000). Our discoveries suggest that specific
131
proteins are also required for this process.
132 133 134
RESULTS
135 136
The esv1 Mutant Has a Low Starch Content at the End of the Night
137 138
To identify plants defective in the control of carbon availability at night, we used a
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forward genetic screen based on an Arabidopsis starvation reporter line (Graf et al.,
140
2010) in which luciferase expression is driven by the promoter of a sugar-repressed
141
gene (At1g10070). Seeds of the reporter line were treated with ethyl methanesulfonate
142
(EMS) to induce point mutations. Then, ten-day-old seedlings of the M2 generation
143
were screened for luciferase-induced bioluminescence at the end of the night, when
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non-mutagenized seedlings of the reporter line showed no bioluminescence. Isolated
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mutant plants were allowed to self-pollinate, and bioluminescence measurements were
146
repeated on M3 plants to check for reproducibility.
147 148
The efficacy of this screen was demonstrated by the identification of two anticipated
149
classes of mutants. Mutants that were shown by iodine staining to contain no starch at
150
the end of the day or the night were not examined further. Such mutants are expected
151
to exhibit symptoms of starvation during the night (Gibon et al., 2006). Mutants that
152
stained reddish-brown rather than blue-black with iodine at the end of the day proved to
153
be defective in ISOAMYLASE1 (ISA1) or ISA2 (Supplemental Figure 1). We showed 5
154
previously that isa1 and isa2 mutants accumulate soluble phytoglycogen (which stains
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reddish-brown with iodine) in place of much of their starch during the day and
156
completely degrade this glucan before the end of the night (Zeeman et al., 1998; Delatte
157
et al., 2005).
158 159
One mutant line exhibited strong bioluminescence at the end of the night, but was
160
shown by iodine staining to have near-normal levels of starch at the end of the day
161
(Figure 1). To test whether the bioluminescence was a consequence of premature
162
exhaustion of starch during the night, rosettes were stained with iodine 2 h before the
163
end of the night. At this point, wild-type rosettes stained a pale color, indicating that
164
some starch remained. Mutant rosettes did not stain, indicating that much less starch
165
remained (Figure 1B). The mutant was named early starvation1, or esv1-1.
166 167
A Splice Site Mutation in At1g42430 Causes the esv1 Phenotype
168 169
The mutation underlying the esv1-1 phenotype was identified by a combination of map-
170
based cloning and whole-genome sequencing. The esv1-1 mutant (Col-0 background)
171
was crossed to a Landsberg erecta (Ler) wild-type plant. F1 plants were allowed to self-
172
pollinate. Seedlings of the F1 generation did not show bioluminescence at the end of
173
the night, but the F2 generation segregated for bioluminescence (Supplemental Figure
174
2A), indicating that the mutation in esv1-1 is recessive. Mapping narrowed the position
175
of the esv1 mutation to a 2.7 Mbp region spanning the centromere of chromosome 1
176
(Supplemental Figure 2, B and C; Supplemental Table 1).
177 178
We used whole genome sequencing to identify the ESV1 locus. DNA from 500 pooled
179
esv1-1 plants in the F2 generation of the mutant x Ler cross was used as a template. In
180
total, 9,000 putative EMS-induced point mutations were identified over the whole
181
genome, two of which were in genes within the target region. One caused an amino
182
acid substitution (glu910lys) in At1g42470, encoding a putative hedgehog receptor
183
located in the plasma membrane. The second was a G-to-A substitution that disrupted
184
the acceptor site at the second intron of At1g42430 (Figure 1C), a gene of unknown 6
185
function. Amplification of full-length cDNA from At1g42430 showed that the mutation
186
results in synthesis of a shorter transcript in esv1-1 than in wild-type plants (Figure 1D).
187
Thus, disruption of the intron splice site may result in splicing at an alternative site and
188
consequent deletions in the transcript. An antiserum was raised to the recombinant
189
protein product of the wild-type At1g42430 cDNA expressed in E. coli. It recognized a
190
protein of the expected mass on immunoblots of extracts of wild-type but not mutant
191
leaves (Figure 1E). The esv1-1 mutant was back-crossed three times to the starvation
192
reporter line before further characterization to minimize the number of unlinked EMS-
193
induced mutations.
194 195
To provide independent evidence that the mutation in At1g42430 was responsible for
196
the early starvation phenotype, we obtained a T-DNA insertion mutant from the GABI-
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Kat (www.gabi-kat.de) collection (Figure 1C). Genotyping confirmed the presence of the
198
T-DNA insert in the fourth exon and immunoblot analysis confirmed that this insertion
199
mutant (named esv1-2) lacked the protein product (Figure 1E). Rosettes of esv1-2
200
showed no staining with iodine 2 h before the end of the night (Figure 1B), which is
201
consistent with the phenotype brought about by the original esv1-1 allele. To evaluate
202
starvation responses in the esv1-2 mutant, we measured transcript levels of two sugar-
203
repressed genes, At3g59940 and At1g76410. These genes encode an F-box protein
204
KMD4 and a C3HC4 type zinc-finger protein ATL8, respectively, and were used to
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monitor starvation responses by Graf et al. (2010).
206 207
Like At1g10070, these genes are expressed at very low levels in wild-type plants at the
208
end of both the day and the night. Their expression rises only when sugar levels are
209
abnormally low, for example during an unexpected extension of the night in wild-type
210
plants, and during the normal night in starchless mutants (Bläsing et al., 2005; Osuna
211
et al., 2007; Usadel et al., 2008; Graf et al., 2010). In esv1-2 mutants, levels of
212
transcript of the two genes at the end of the day were similar to those in wild-type
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plants, but levels at the end of the night were considerably elevated (Supplemental
214
Figure 3).
215 7
216
ESV1 is Similar to a Second Predicted Arabidopsis Protein; Both Are Conserved
217
Throughout the Plant Kingdom
218 219
The predicted ESV1 protein is composed of 426 amino acids, with a predicted mass of
220
about 49 kDa. It contains no previously-annotated domains but has a proline-rich region
221
at the C-terminal end (11 proline residues between amino acids 397 and 425) and is
222
enriched in tryptophan and other aromatic amino acid residues in the C-terminal two-
223
thirds of the protein (~11% of the amino acid residues between amino acids 130 and
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380 are tryptophans). Within this region are motifs in which single or paired aromatic
225
amino acids are separated by two or three other amino acids, usually including one or
226
more acidic residues (E or D), for example WWETW, WTDKW, WEETWW,
227
WYEKWWEKY, WWEKWGEHY (Figure 2, Supplemental Figure 4).
228
A BLAST search (http://www.arabidopsis.org/Blast/index.jsp) showed that an
229
unannotated protein encoded by At3g55760 has the highest sequence similarity to
230
ESv1: the predicted amino acid sequences are 38% identical. We refer to this second
231
protein as LIKE ESV1, or LESV. It is composed of 578 amino acids with a predicted
232
mass of 66 kDa. LESV1 lacks the proline-rich C-terminal region of ESV1 but shares the
233
tryptophan-rich region with aromatic motifs similar to those in ESV1. Within this region,
234
the two proteins have similar numbers of tryptophan residues and the highest level of
235
overall identity (Figure 2A, Supplemental Figure 4A). Unlike ESV1, LESV is represented
236
on commonly-used Arabidopsis microarrays. Examination of publicly available
237
microarray data revealed that LESV is expressed in all organs of the plant. In leaves,
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transcript levels are high at the end of the night and low during much of the day
239
(Supplemental Figure 5). LESV is strongly co-expressed with several genes encoding
240
enzymes of starch metabolism: the top twenty co-expressed genes from Atted-II
241
(www.atted.jp: Obayashi et al., 2009) encode enzymes including ISOAMYLASE3
242
(ISA3), α-GLUCAN PHOSPHORYLASE2 (PHS2), GLUCAN, WATER DIKINASE (GWD,
243
also known as STARCH EXCESS1, SEX1), STARCH-EXCESS 4 (SEX4),
244
DISPROPORTIONATING ENZYME1 (DPE1) and DPE2, STARCH BRANCHING
245
ENZYME3 (BE3), α-AMYLASE3 (AMY3) and ADP-GLUCOSE
8
246
PYROPHOSPHORYLASE LARGE SUBUNIT4 (APL4) [Supplemental Table 2; see
247
Smith (2012) for a discussion of the roles of these enzymes].
248 249
Genes homologous to ESV1 and LESV are found in land plants and green algae,
250
including microalgae in the genus Ostreococcus which have the smallest known
251
genomes among eukaryotes (Supplemental Figure 4B, Supplemental Table 3,
252
Supplemental Data Set 1). The genes are apparently absent from prokaryotes, red
253
algae, and other eukaryotic life-forms. ESV1 was included in a phylogenomic inventory
254
of proteins specific to the plant lineage (GreenCut2: Karpowicz et al., 2011). The high
255
level of evolutionary conservation of ESV1 and LESV protein sequences suggests that
256
both proteins have important functions that are specific to the Viridiplantae.
257 258
ESV1 and LESV are Chloroplastic and Bind to Starch Granules
259 260
Chloroplastic locations for ESV1 and LESV were suggested by previous publications on
261
the chloroplast proteome [e.g. Bayer et al. (2011) for ESV1; Kleffmann et al. (2004),
262
Peltier et al. (2006) for LESV]. The ChloroP program predicts a 56-amino acid N-
263
terminal chloroplast transit peptide (cTP) for LESV but no cTP for ESV1. However, the
264
predicted proteins most similar to ESV1 from Glycine max, Prunus persica, Vitis
265
vinifera, Manihot esculenta and Populus trichocarpa have putative transit peptides. We
266
sought to confirm the plastidial localisation of the Arabidopsis proteins by transiently
267
expressing them in leaves of woodland tobacco (Nicotiana sylvestris) as C-terminal
268
fusions to Yellow Fluorescent Protein (YFP). For both proteins, YFP fluorescence was
269
exclusively located in chloroplasts and was associated with discrete bodies likely to be
270
starch granules (Figure 2B). Stable expression of these constructs in Arabidopsis gave
271
the same result (Supplemental Figure 6A). To provide more information about the
272
location of the proteins within chloroplasts, we also expressed the fusion proteins
273
transiently in the pgm mutant of N. sylvestris, which lacks chloroplastic
274
phosphoglucomutase and therefore cannot synthesize starch (Hanson and McHale,
275
1988). In the pgm mutant, YFP appeared as a diffuse signal in the chloroplast stroma
276
for both fusion proteins rather than being associated with discrete structures. This 9
277
suggests that the fluorescent structures in chloroplasts of wild-type N. sylvestris are
278
indeed starch granules.
279 280
ESV1 and LESV were both partitioned between the soluble and insoluble (starch-
281
containing) fraction of leaves. Immunoblotting of extracts of wild-type N. sylvestris
282
leaves transiently expressing ESV1 or LESV as a YFP fusion revealed that the fusion
283
proteins were more abundant in the insoluble fraction – the expected location for starch-
284
bound proteins - than in the soluble fraction. Both proteins were mostly soluble when
285
transiently expressed in pgm leaves, which lack starch (Supplemental Figure 6B). In
286
wild-type Arabidopsis leaves, ESV1 and LESV proteins were also present in both
287
insoluble and soluble fractions (Figure 2C). The proteins were largely soluble at the end
288
of the night when starch levels were low, and they were largely insoluble at the end of
289
the day when starch level were maximal. ESV1 and LESV were present in proteins
290
extracted from purified starch from wild-type Arabidopsis leaves and were among the
291
most abundant granule-bound proteins in these leaves (Figure 2D). As the ESV1 and
292
LESV genes are conserved throughout the plant kingdom, we examined whether the
293
proteins are present in starches from species of economic significance. Both proteins
294
were present in starch purified from roots of cassava, tubers of potato, and mature
295
grains of rice and maize (Supplemental Table 4).
296 297
esv1 Mutants Cannot Adjust the Rate of Starch Degradation to the Length of the
298
Night
299 300
To characterize the defect in starch degradation in the esv1 mutants, we analyzed
301
starch contents in rosettes during a normal 12-h night and in rosettes subjected to
302
darkness after only 8 h of light (an early night) (Figure 3). Under both conditions, as
303
expected, wild-type plants degraded starch at a near linear rate and did not exhaust
304
their reserves before the end of the night. The rates of starch degradation were faster in
305
esv1 mutant plants than in wild-type plants, such that starch reserves were completely
306
exhausted 2 h before the end of the night in normal nights and 4 h before the end of the
307
night in early nights (Figure 3, A to C). Extrapolation of the near-linear phase of starch 10
308
degradation for esv1 plants, over the first 8 h of the night, revealed that these rates
309
(estimated from linear regression analysis) would exhaust starch reserves after
310
approximately 9 h in both normal (12-h) nights and early nights. Thus, wild-type plants
311
adjusted the rate of starch degradation to the length of the night, but esv1 mutant plants
312
failed to make this adjustment correctly.
313 314
To provide more information about the alterations in the control of starch degradation in
315
esv1 plants, we subjected batches of esv1 and wild-type plants to different light levels
316
over a single day to generate plants with very different starch levels at the start of the
317
12-h night. As expected, the rate of starch degradation in wild-type plants was adjusted
318
such that the starch reserves lasted for 12 h over a wide range of end-of-day starch
319
contents. In esv1 plants, the time at which starch reserves were exhausted depended
320
upon the initial starch content. Reserves were exhausted after 9 h of darkness in plants
321
with 7.5 mg starch g-1 fresh weight at the end of the day, and after 6 h of darkness in
322
plants with 2 mg starch g-1 fresh weight at the end of the day (Supplemental Figure 7, A
323
and B). Whereas in wild-type plants, the relative rate of starch degradation (fraction of
324
end-of-day starch degraded per unit time) was independent of starch content, in esv1
325
plants, the relative rate decreased as starch content decreased (Supplemental Figure
326
7C). Thus, in esv1, the dependence of starch degradation on time until dawn has
327
decreased or been lost.
328
In most experiments, esv1 mutants had lower rates of net starch accumulation than
329
wild-type plants during the day and hence, lower starch contents at the onset of
330
darkness (Figure 3). Interestingly, the levels of both sucrose and maltose in esv1-2
331
plants were higher than in wild-type plants during the day but lower than in wild-type
332
plants towards the end of the night (Figure 3D and E). Elevation of sucrose levels during
333
the day is commonly observed in Arabidopsis mutants with defects in pathways of
334
starch biosynthesis or degradation (e.g. Chia et al., 2004; Comparot-Moss et al., 2010;
335
Mugford et al., 2014). Elevated levels of maltose are generally indicative of starch
336
degradation (Weise et al., 2004 ). Taken together, these results indicate that esv1
337
mutants usually accumulate less starch than wild-type plants because they degrade
338
starch during the day as well as during the night. The low levels of sucrose and maltose 11
339
towards the end of night in esv1 are consistent with the observed premature starch
340
depletion in these plants.
341 342
We investigated whether loss of LESV also affects starch turnover by examining two T-
343
DNA insertion mutants (Figure 1C). The absence of LESV protein from these mutants
344
was confirmed by immunoblotting with a specific antiserum (Figure 1E). Diel patterns of
345
starch accumulation and loss in the lesv mutants were generally similar to those of wild-
346
type plants (Figure 3, A, F and G). Sucrose and maltose levels in the lesv-1 mutant
347
were also similar to those of wild-type plants over the day-night cycle (Figure 3, D and
348
E). Starch turnover and sucrose and maltose levels throughout the day-night cycle in an
349
esv1-2 lesv-1 double mutant resembled those of the esv1 parent (Figure 3, A, D and E).
350 351
Lack of ESV1 Affects Starch Contents in Non-Photosynthetic Tissues
352 353
We investigated whether ESV1 is important for the control of starch content in non-
354
photosynthetic parts of the plant (Figure 4). Detailed examination of iodine-stained
355
leaves showed that cells immediately adjacent to veins accumulate less starch during
356
the day in esv1 mutants than in wild-type plants. This netted pattern of starch
357
accumulation in esv1 leaves was particularly pronounced after an extended light period
358
of 24 h (Figure 4A). Staining for starch also revealed lower starch contents in esv1 than
359
in wild-type plants in columella cells of root caps, stems, flowers and siliques (Figure 4,
360
B to D).
361
Plants lacking ESV1 had several developmental and morphological phenotypes that
362
may be attributable to defective starch metabolism. After 24 d of growth, rosettes of
363
esv1 plants were smaller than those of wild-type plants (Figure 4E), and their fresh
364
weights were about 40% lower. Mutant plants also flowered later than wild-type plants.
365
Other mutants with defects in diel starch turnover in leaves are slow-growing in 12 h
366
light, 12 h dark cycles, e.g., the essentially starchless mutants pgm and adg1 (lacking
367
the small subunit of ADPglucose pyrophosphorylase) and the starch-degradation
368
mutants sex1 (lacking glucan, water dikinase) as well as bam1 bam3 (lacking β-
369
amylases 1 and 3) (Caspar et al., 1985; Lin et al., 1988; Yu et al., 2001; Fulton et al., 12
370
2008), and some exhibit delayed flowering, e.g., sex1 and pgi (lacking plastidial
371
phosphoglucomutase) (Eimert et al., 1995; Corbesier et al., 1998; Yu et al., 2000). The
372
axillary shoots of esv1 flower stems grew with a wider angle from the main stem than
373
those of wild-type plants (Figure 4F). Wide stem angles are characteristic of mutants
374
defective in the stem gravitropic response, which is dependent on the presence of
375
starch granules in endodermal cells (Weise and Kiss, 1999; Fujihira et al., 2000;
376
Tanimoto et al., 2008). Whereas the endodermis of wild-type and lesv stems contained
377
large starch granules, no starch was visible in this tissue in esv1 stems (Figure 4D).
378 379
Over-Expression of ESV1 and LESV Proteins Alters Leaf Starch Content
380 381
To provide more insight into the importance of ESV1 and LESV proteins in controlling
382
starch content in leaves, we generated plants with elevated amounts of these proteins
383
by constitutively expressing them as YFP fusions (Supplemental Fig 8A). Expression of
384
ESV1-YFP in either wild-type or esv1 mutant plants resulted in elevated starch
385
contents. In some lines, starch content was elevated at the end of the night but not at
386
the end of the day, whereas in the line with the highest level of expression of ESV1-YFP
387
(line 3-2), starch content was strongly elevated throughout the day-night cycle (Figure
388
3G).
389 390
We also generated plants in which ESV1 protein without a tag was expressed in an
391
esv1 background. These plants had smaller increases in ESV1 protein than those
392
expressing ESV1-YFP (Supplemental Figure 8B). Nonetheless, as for plants expressing
393
ESV1-YFP, starch contents of leaves were elevated relative to those of wild-type plants
394
at both the end of the day and the end of the night (Supplemental Figure 8C), showing
395
that this phenotype was not an artefact arising from the use of a YFP fusion protein.
396
Given this striking effect of ESV1 over-expression, we examined whether LESV over-
397
expression affected starch content. Constitutive expression of LESV-YFP in a wild-type
398
background had little impact on starch content at the end of the day, but starch content
399
10 h into the night and at the end of the night was about three-fold lower than in wild-
400
type plants (Figure 3G, Supplemental Figure 8A). These data suggested that LESV13
401
overexpressing plants might be starving at the end of the night. Consistent with this
402
expectation, the transcript abundance of two starvation genes was higher in LESV-
403
overexpressing plants than in wild-type plants at the end of the night, and was similar to
404
that in esv1 plants at this point (Supplemental Figure 3).
405 406
Altered Levels of ESV1 and LESV Affect Starch Granule Morphology, Number and
407
Composition but Have Only Minor Effects on Amylopectin Structure
408
Starch granules from leaves of esv1 and lesv mutants and the double mutant esv1 lesv
409
were roughly discoid in shape, like those of wild-type leaves. However, granules of the
410
mutants were less regular in shape than wild-type granules: they consistently had lobed
411
or wavy outlines (Figure 5). ESV1 over-expressing plants had thicker, larger granules
412
than wild-type plants. LESV over-expressing plants had many more granules per
413
chloroplast than wild-type plants. These granules were highly variable in size and
414
shape, and many were much smaller than granules of wild-type plants.
415 416
The starch of both esv1 and lesv mutants consistently contained about 60% more
417
amylose than that of wild-type plants (Figure 6). It seemed likely that this change in
418
starch composition was a secondary rather than the primary consequence of the
419
mutations, since the esv1 and lesv mutations have the same effect on amylose content
420
but radically different effects on the rate of starch degradation. To investigate this
421
further, we examined whether loss of amylose affects starch degradation in esv1
422
mutants by generating a double mutant lacking both ESV1 and granule-bound starch
423
synthase (GBSS), the enzyme responsible for amylose biosynthesis (Seung et al.,
424
2015). As expected, the esv1 gbss double mutant lacked amylose. Its starch content
425
was lower than that of wild-type or gbss plants and comparable to that of esv1 at both
426
the end of the day and two hours before the end of the night (Supplemental Figure 9A).
427
Thus, loss of ESV1 results in faster starch degradation regardless of whether amylose
428
is present in the starch.
429 430
Over-expression of ESV1 and LESV had very different effects on starch composition. In
431
ESV1-over-expressing plants, amylose content was similar to that of esv1 mutants – 14
432
about 60% higher than in wild-type plants. Over-expression of LESV drastically reduced
433
amylose content (Figure 6A; Supplemental Figure 9B). The variation in amylose content
434
caused by changes in amounts of ESV1 or LESV was not attributable to variation in the
435
amount of GBSS in the starch. There was no obvious relationship between GBSS
436
protein levels and amylose levels in plants with different levels of ESV1 or LESV
437
proteins (Supplemental Figure 9C). Loss or over-expression of ESV1 caused only minor
438
alterations in the levels of starch-bound phosphate. Changes in LESV content had
439
greater effects: loss of LESV reduced starch phosphate content by about 25%, whereas
440
over-expression increased it by about 75% (Supplemental Figure 9D).
441 442
We investigated whether altered levels of ESV1 or LESV proteins affect the structure of
443
the major starch polymer amylopectin. The chain length distribution of amylopectin
444
differed slightly between esv1, lesv, ESV1 and LESV over-expressing lines and wild-
445
type starch (Figure 6B), but these differences were small in comparison to those seen in
446
Arabidopsis mutants deficient in enzymes of starch biosynthesis (e.g. Pfister et al.,
447
2014). The chain length distributions of the β-limit dextrin of amylopectin from esv1 and
448
lesv mutants were also similar to those of wild-type plants (Supplemental Figure 9F).
449
These results indicate that neither chain elongation nor branching/debranching during
450
amylopectin biosynthesis are strongly affected by the loss of ESV proteins.
451 452
Loss of ESV1 Modifies but Does Not Abolish the Effects of Mutations That
453
Reduce Starch Degradation
454 455
We considered the possibility that ESV1 interacts with a specific enzyme of starch
456
degradation and inhibits its activity. To test this idea, we created a series of double
457
mutant plants that lacked both ESV1 and a specific enzyme shown previously to be
458
involved in starch degradation in the chloroplast at night (Figure 7). Interpretation of the
459
phenotypes of mutants lacking enzymes of starch degradation is complicated by
460
redundancy within the pathway: for most enzymes, loss does not prevent starch
461
degradation but rather reduces the proportion of end-of-day starch that is consumed
462
during the night (Scialdone et al., 2013). Nonetheless, if ESV1 inhibits one specific 15
463
enzyme of starch degradation, the loss of both this target enzyme and ESV1 should
464
give a starch turnover phenotype identical to that given by the loss of the target enzyme
465
alone. The loss of both ESV and any enzyme other than its direct target might be
466
expected to give a phenotype different from that of either parent mutant.
467 468
We first investigated the effect of losing ESV1 on the phenotype of the sex1 mutant,
469
which lacks GWD . This enzyme catalyzes the first step in degradation: the
470
phosphorylation of the C6-position of glucose residues in amylopectin (Yu et al., 2001;
471
Ritte et al., 2002). Starch degradation in sex1 mutants is severely impaired and starch
472
accumulates to high levels. It is thought that phosphorylation at the granule surface by
473
GWD disrupts the organization of the starch polymers and facilitates degradation by
474
starch-hydrolyzing enzymes (Blennow and Engelsen, 2010; Hejazi et al., 2008; Hejazi
475
et al., 2009; Hejazi et al., 2010). The esv1 sex1 mutants had leaf starch contents that
476
were different from those of either parent. Starch contents were significantly lower than
477
those of the sex1 mutant, but much higher than those of esv1 and wild-type plants.
478
(Figure 7A, Supplemental Figure 9F). However, the starch phenotype of the double
479
mutant was very different in other parts of the plant. Whereas sex1 mutants accumulate
480
very high levels of starch throughout the plant, including root caps (Caspar et al., 1991;
481
Yu et al., 2001), the esv1 sex1 mutant had very low levels of starch in root cap cells,
482
similar to the esv1 parent (Figure 4B). The esv1 sex1 mutant also had little starch in the
483
major veins and petioles of mature leaves and around the meristem. These regions had
484
very high starch contents in the sex1 parent (Figure 7B).
485 486
As for the esv1 sex1 mutant, leaves of other double mutants lacking both ESV1 and a
487
protein necessary for starch degradation had starch turnover phenotypes different from
488
those of their parents (Figure 7, C and D). Loss of ESV1 reduced the severity of the
489
starch-excess phenotype in all mutant backgrounds examined, but double mutants
490
retained more starch in the rosette at the end of the night than esv1 and wild-type
491
plants. The starch contents of leaves of the esv1 sex4 and esv1 pwd double mutants
492
were reduced relative to that of sex4 (lacking a glucan phosphate phosphatase: Kötting
493
et al., 2009) and pwd (lacking phosphoglucan water dikinase: Baunsgaard et al., 2005; 16
494
Kötting et al., 2005) by about one third or less and were always greatly in excess of the
495
starch content of wild-type plants. Mutants lacking ESV1 and either the major
496
chloroplastic β-amylase BAM3, the β-amylase-like protein BAM4 (Fulton et al., 2008) or
497
the glucan phosphate phosphatase-like protein LSF1 (Comparot-Moss et al., 2010) had
498
starch contents that were reduced by 50% or more relative to their starch excess parent
499
at the end of the day and the end of the night. Because loss of ESV1 reduced the
500
severity of the starch excess phenotype in all of the starch degradation mutants, we
501
suggest that its role in starch degradation is not primarily as a negative regulator of any
502
one of the five starch-degradation proteins affected in the mutants. However, these
503
results must be interpreted with caution because it remains possible that – as for sex1 -
504
the impact of loss of ESV1 on the starch-excess phenotypes is different in
505
photosynthetic and non-photosynthetic cells of the rosette.
506 507
We also investigated whether the high rate of starch degradation in the esV1 mutant
508
results from activation of a degradative enzyme that is redundant with respect to starch
509
degradation in wild-type plants. Accordingly, we crossed esV1-1 with mutants lacking
510
either the β-amylase BAM1 or the α-amylase AMY3. Loss of either of these
511
chloroplastic proteins has no effect on starch turnover in a wild-type background (Yu et
512
al., 2005; Fulton et al., 2008). We found that the esV1 bam1 and esV1 amy3 double
513
mutants had the same very low starch contents at the end of the night as the esV1
514
parent (Figure 7, C and E); thus, the high rate of starch degradation in esv1 does not
515
require the activity of either BAM1 or AMY3.
516 517
DISCUSSION
518 519
This work describes two proteins with central and previously unsuspected roles in plant
520
carbohydrate metabolism. We show that the presence and/or levels of the ESV1 and
521
LESV proteins are crucial for normal patterns of starch biosynthesis and degradation in
522
the Arabidopsis plant, and hence for normal growth and productivity. Our results lead us
523
to propose that both proteins are involved in determining the conformation of the starch
524
granule matrix, and that the perturbations of starch turnover in plants with altered levels 17
525
of the proteins are indirect consequences of abnormal starch granule structures. Below
526
we present the evidence for this proposal and discuss the wider significance of our
527
findings for the understanding of carbon storage and allocation in plants.
528 529
ESV1 and LESV Are Important for Normal Starch Turnover in Leaves
530 531
Our starvation reporter screen identified ESV1 as a protein required for normal rates of
532
starch degradation in Arabidopsis leaves at night. Whereas in wild-type plants, starch
533
reserves are used at an essentially linear rate at night such that they are exhausted
534
almost exactly at dawn, in the absence of ESV1, starch reserves are consumed in a
535
non-linear manner and are exhausted prior to dawn. Accelerated starch degradation at
536
night was previously reported in the double mutant cca1 lhy, which lacks two MYB
537
transcription factors that are central components of the circadian clock and thus
538
anticipates dawn several hours too early (Graf et al., 2010). It seems highly unlikely that
539
the rapid starch degradation in esv1 mutants is brought about by a defective circadian
540
clock. The mutant does not exhibit short-period phenotypes such as early flowering, and
541
the ESV1 protein is located in the chloroplast whereas known clock components are
542
nuclear and cytosolic.
543 544
The esv1 phenotype is similar in several respects to that of isa mutants. The isa1 and
545
isa2 mutants also exhaust their glucan reserves prior to dawn and appear to degrade
546
some storage glucan during the day as well as at night (Delatte et al., 2005). The
547
reserve glucans in these mutants are largely soluble rather than in granular form
548
(Zeeman et al., 1998; Delatte et al., 2005). Loss of normal control of degradation is
549
expected in this case. Our chemical kinetic models capable of explaining the
550
dependency of starch degradation on starch content and time until dawn require the
551
interaction of controlling molecules with the solid surface of the starch granule, and they
552
predict that control will be lost if glucans are soluble (Scialdone et al., 2013). esv1
553
mutants accumulate glucans as starch granules, so loss of control of glucan
554
degradation at night is not due to a radical change in glucan solubility. However, the
555
loss of control may be generally analogous to that in isa mutants in that it may stem 18
556
from increased accessibility of glucans to starch-degrading enzymes. We discuss this
557
possibility below.
558 559
LESV is less important for normal starch turnover than ESV1. The pattern of starch
560
turnover in the lesv mutant is similar to that of wild-type plants. The involvement of
561
LEST in processes underpinning normal starch metabolism is apparent from the
562
significantly altered levels of amylose and of granule-bound phosphate in the lesv
563
mutant and from the effects of LESV over-expression in Arabidopsis leaves. As with
564
esv1 mutants, plants with elevated LESV consume starch too rapidly during the night so
565
that reserves are exhausted before dawn. By contrast, over-expression of ESV1 results
566
in very high starch levels throughout the day-night cycle. Overall, our results show that
567
control of ESV1 and LESV protein levels is essential for normal starch turnover in
568
Arabidopsis leaves.
569 570
The ESV1 and LESV Proteins Are Directly Associated with Starch Granules
571 572
Experiments employing fluorescently-tagged proteins confirmed findings from previous
573
proteomic studies that both ESV1 and LESV proteins are located inside the chloroplast.
574
Together with cell fractionations, these experiments revealed that both proteins are at
575
least in part associated with starch granules. Changes during the day-night cycle in the
576
fraction of the proteins that is granule bound suggest that both are incorporated into
577
granules as they grow during the day and are then released as they are degraded at
578
night. Although LESV transcript levels vary several-fold over the day-night cycle, being
579
low at the end of the day and high at the end of the night, the total amount of LESV
580
protein is similar at these two time points. Thus, it appears that large daily changes in
581
the transcript level of LESV are not reflected at the protein level, a feature that LESV
582
shares with several other proteins of starch metabolism (Skeffington et al., 2014).
583
Equivalent information is not available for ESV1 because it is not represented on
584
commonly-used Arabidopsis microarrays. Most of the proteins previously reported to be
585
present in both starch granules and the stroma are enzymes of starch biosynthesis and
586
degradation or proteins that interact directly with these enzymes. They include various 19
587
isoforms of starch synthase and starch-branching enzyme (e.g. Denyer et al., 1993; Mu-
588
Forster et al., 1995; Grimaud et al., 2008) and the enzymes responsible for
589
phosphorylation and dephosphorylation of the starch granule surface (e.g. Yu et al.,
590
2001; Kötting et al., 2009; Santelia et al., 2011). The dual location of ESV1 and LESV
591
proteins in the stroma and the starch granule thus indicates a direct role in the
592
biosynthesis, assembly and/or degradation of starch granules.
593 594
Proteins that bind to starch usually possess either Carbohydrate Binding Modules
595
(CBMs) or Surface binding Sites (SBSs) or both. Arabidopsis proteins with one or more
596
of these domains include starch synthases, starch branching and debranching
597
enzymes, amylases, enzymes that phosphorylate or dephosphorylate the starch granule
598
surface, and the recently-characterized PTST (PROTEIN TARGETING TO STARCH)
599
protein that facilitates the binding to starch of granule-bound starch synthase (e.g.
600
Palopoli et al., 2006; Glaring et al., 2011; Meekins et al., 2013; Seung et al., 2015).
601
Starch-binding CBMs fall into distinct classes, for example, GWD and AMY3 both
602
possess two CBM45 domains in tandem and isoamylases, branching enzymes and
603
PTST possess CBM48 domains. SBSs are generally highly variable and require
604
experimental definition (e.g. Cockburn et al., 2013; Meekins et al., 2014). Neither ESV
605
nor LESV possesses a recognizable CBM, and further work will be required to discover
606
whether they possess SBSs. However, the observation that both proteins contain a
607
large domain strongly enriched in conserved tryptophan residues, and with conserved
608
phenylalanine and tyrosine resides, leads us to speculate that they may be capable of
609
binding to specific glucan structures. Known starch-binding CBMs are 90-130 amino
610
acids long and typically have four conserved aromatic amino acid residues that align the
611
protein with the non-polar faces of glucose residues in starch chains (Christiansen et al.,
612
2009). Although they are often found singly, CBMs also occur in tandem – for example
613
STARCH SYNTHASE3 from Arabidopsis has three CBMs (Palopoli et al., 2006). The
614
tryptophan-rich regions of ESV1 and LESV contain over 35 conserved aromatic amino
615
acid residues arranged in motifs. It seems possible that the repeated motifs may
616
mediate binding to numerous glucans or facilitate interaction with long glucans at
617
numerous contact points. 20
618 619
ESV1 Probably Acts Upstream of Enzymes of Starch Degradation
620 621
To discover whether ESV1 might inhibit the action of a protein required for starch
622
degradation, we examined the effect of loss of ESV1 on the phenotypes of starch-
623
excess mutants lacking individual proteins involved in starch degradation. Loss of ESV1
624
reduced but did not abolish the starch excess phenotypes of the sex1, sex4, lsf1, bam3
625
and bam4 mutants. Loss of ESV1 from plants lacking AMY3 or BAM1 - starch-
626
degrading enzymes that are largely redundant in a wild-type background (Yu et al.,
627
2005; Fulton et al., 2008) - resulted in esv1-like phenotypes. The pathway of starch
628
degradation is not amenable to straightforward genetic analysis because of multiple
629
redundancies. Nonetheless, these results allow us to make three proposals about the
630
role of ESV1 as follows.
631 632
First, ESV1 is unlikely to act through direct inhibition of SEX1, PWD, SEX4, LSF1,
633
BAM3 or BAM4. Second, the accelerated rate of starch degradation in the esv1 mutant
634
is largely catalyzed by the same enzymes and proteins responsible for starch
635
degradation in wild-type plants. SEX1, SEX4, LSF1, BAM3 and BAM4 are all required
636
for the accelerated rate of degradation in the esv1 mutant, and we found no evidence
637
that this accelerated rate requires either of the normally “redundant” enzymes, AMY3
638
and BAM1. Third, in mesophyll cells, ESV1 may act at or prior to the start of the
639
pathway of starch degradation. Loss of ESV1 affected the starch excess phenotypes of
640
mutants lacking both downstream and initial enzymes of the pathway. The observation
641
that it had smaller effects on the starch excess phenotypes of sex1, pwd and sex4 than
642
on those of mutants lacking β-amylases could indicate that ESV1 function is linked more
643
closely to the phosphorylation/dephosphorylation of the granule surface than to the
644
actions of downstream hydrolytic enzymes. However, as discussed above, there is no
645
evidence that ESV1 directly inhibits the enzymes of phosphorylation/dephosphorylation,
646
and its loss and overexpression have only small effects on levels of starch-bound
647
phosphate.
648 21
649
ESV1 May Play a Role in Determining the Organization of Glucans in the Granule
650
Matrix
651 652
Given that ESV1 binds to starch granules and appears to act upstream of the pathway
653
of starch degradation, it seems possible that it influences the accessibility of glucans
654
within the matrix to proteins involved in starch degradation. Any such influence is
655
unlikely to be due to a direct effect of ESV1 on starch polymer composition or structure.
656
Loss or over-expression of either ESV1 or LESV affected the amylose content of starch,
657
and had minor effects on amylopectin chain length distribution. However, these changes
658
are highly unlikely to account for the accelerated rate of starch degradation in the esv1
659
mutant. First, both esv1 and lesv mutants have elevated amylose contents, but only the
660
esv1 mutant has accelerated starch degradation. Second, abolition of amylose from the
661
esv1 mutant (in the esv1 gbss mutant) does not alter its starch degradation phenotype.
662
Third, the alterations in chain length distribution of amylopectin are much smaller than in
663
many mutants that do not exhibit accelerated starch degradation (e.g. starch synthase
664
mutants: Szydlowski et al., 2009; Pfister et al., 2014).
665 666
Our data indicate that ESV1 and LESV directly influence the organization of the starch
667
granule matrix rather than the biosynthesis of the starch polymers that comprise it. The
668
esv1 and lesv mutants have irregular granule shapes, and over-expression of ESV1 and
669
LESV dramatically alters granule size, morphology and - in the case of LESV –
670
abundance. ESV1 over-expressers have large, thick granules, whereas LESV over-
671
expressers have increased numbers of granules of very variable size and shape. These
672
effects are unlikely to be due to altered amylopectin structure in the over-expressing
673
lines: the alterations in amylopectin structure are minor compared with those of other
674
starch mutants that retain normal granule shapes and numbers.
675 676
Little is known about how starch polymers are organized to form the starch granule
677
matrix. Waigh and colleagues (Waigh et al., 1998; Waigh et al., 2000) showed that
678
many physical properties of starch – including gelatinization, freezing/thawing and
679
hydration/dehydration - can be explained by modelling it as a chiral side-chain liquid 22
680
crystalline polymer, a self-organizing structure. By extrapolation, and in the absence of
681
a biological explanation, it has been assumed that starch chains synthesized at the
682
granule surface in vivo assemble into double helices that self-organize through physical
683
processes, forming the semi-crystalline lamellar structure of the matrix. The discovery of
684
the ESV1 and LESV proteins raises the possibility that correct assembly of the matrix
685
involves proteins that bind to starch polymers, in addition to physical processes.
686 687
A direct role for ESV1 in the correct organization of the granule matrix could explain why
688
its loss perturbs the rate of starch degradation. We suggest that the degree to which
689
hydrolytic enzymes can access starch polymers is in part determined by ESV1. By
690
helping to confer a high level of matrix organization, ESV1 could restrict access by
691
hydrolytic enzymes. If, in the absence of ESV1, the matrix is less organized, it could be
692
much more susceptible to degradation by hydrolytic enzymes than in wild-type plants.
693
In wild-type plants, control over the rate of starch degradation may be exerted via
694
modulation of the level of starch phosphorylation. Phosphorylation of glucose residues
695
at the granule surface by GWD and PWD is thought to interfere with the regular packing
696
of double helices, thus rendering the starch polymers more accessible to hydrolytic
697
enzymes (Blennow and Engelsen, 2010; Hejazi et al., 2008; Hejazi et al., 2009; Hejazi
698
et al., 2010). We recently presented evidence that phosphorylation may be the point at
699
which circadian clock control over starch degradation is exerted. In the absence of
700
PWD, the rate of starch degradation could no longer be correctly adjusted to the length
701
of the night (Scialdone et al., 2013). It seems plausible that an altered level of starch
702
matrix organization in esv1 mutants may render the modulation of phosphorylating
703
activities less effective as a means of controlling the rate of degradation.
704 705
Although there is no clear starch turnover phenotype in the lesv mutant, the altered
706
amylose and starch phosphate contents of the mutant starch and the large effects of
707
LESV over-expression on starch degradation and granule size, shape and number
708
indicate that LESV too may play a role in matrix organization and in modulating the
709
effects of phosphorylation on the accessibility of starch polymers to hydrolytic enzymes.
710
However ESV1 and LESV appear to have opposite roles in these respects, ESV1 23
711
promoting a high level of organization of the granule matrix and LESV potentially
712
reducing the level of organization. These differences in function may stem from
713
divergent features of the protein sequences. The two proteins share a tryptophan-rich
714
domain, in which many of the aromatic amino acid residues are contained within distinct
715
motifs. In ESV1 sequences, there are 39 conserved aromatic residues within this
716
domain. LESV sequences contain 37 conserved aromatic residues in the same region,
717
30 of which are in the same amino acid position as in ESV1 (Supplemental Figure 4A).
718
However, motif sequences differ between the two proteins. Comparisons of ESV1 and
719
LESV proteins from a wide range of starch-synthesizing organisms shows that many
720
motifs in the domain are conserved within either ESV1 or LESV but not shared between
721
them. ESV1 also possesses a proline-rich terminal domain that is absent from LESV,
722
and LESV has a unique >100-amino-acid N-terminal domain. Further information about
723
the roles of these proteins will come from investigation of the starch- and glucan-binding
724
properties of wild-type and mutant forms combined with detailed physical analyses of
725
starches from plants expressing wild-type and mutant forms at different levels.
726 727
The Importance of ESV1 and LESV for Starch Degradation Varies between Organs
728 729
Examination of the esv1 mutant indicates that the importance of the ESV1 protein for
730
normal starch turnover varies from one organ and tissue to another. In leaves, loss of
731
ESV1 accelerates starch degradation and also reduces the extent of starch
732
accumulation during the day. The reduction in starch accumulation may be due to the
733
occurrence of starch degradation in the light as well as in the dark: the accumulation of
734
maltose – the main product of starch degradation in the chloroplast (Weise et al., 2004)
735
- in esv1 leaves during the day supports this idea. Thus, loss of ESV1 may render leaf
736
starch granules partially accessible to hydrolytic enzymes during the day, as well as
737
increasing access for these enzymes during the night.
738 739
In other organs of the plant, loss of ESV1 has a more radical effect on starch turnover.
740
The starch content of root columella cells and of stems in esv1 plants is very much
741
lower in mutant than in wild-type plants. In sex1 mutant plants, loss of ESV1 has little 24
742
effect on the starch excess phenotype in mesophyll cells of the leaf but abolishes starch
743
accumulation in root cap, vein and petiole cells. These observations indicate that the
744
importance of ESV1 for normal starch metabolism is dependent on the dynamics of
745
starch turnover. In wild-type leaves, little or no starch degradation occurs during most of
746
the light period: the rate of starch biosynthesis and starch accumulation are the same.
747
In the absence of ESV1, some degradation occurs during the day, but this rate is far
748
lower than the rate of biosynthesis. In other organs of the plant, starch may be subject
749
to simultaneous biosynthesis and degradation at all times. In the embryo, for example,
750
the effects of loss of starch degrading enzymes on starch content in early development
751
reveal that starch biosynthesis and degradation occur simultaneously throughout the
752
period of accumulation and loss of starch (Andriotis et al., 2010). Thus, we anticipate
753
that loss of ESV1 effectively prevents starch storage in organs other than the leaf
754
because it permits faster degradation without a change in the rate of biosynthesis.
755 756
Are ESV1 and LESV part of the control mechanism that adjusts the rate of leaf starch
757
degradation according to time of dawn and starch content? Such a role cannot be ruled
758
out, but at present there is no evidence that the actions of these proteins are subject to
759
either transcriptional or post-translational control on a short-term basis. The wide
760
occurrence and conservation of both proteins, and the importance of ESV1 in many
761
organs of the plant, indicate that they are fundamental components of the starch
762
biosynthesis and turnover machinery in general.
763 764
METHODS
765 766
Plant Materials and Growth Conditions
767 768
All Arabidopsis thaliana mutants were in the Col-0 background. Starch-excess mutants
769
were described previously (amy3, Yu et al., 2005; bam mutants, Fulton et al., 2008;
770
gbss, Seung et al., 2015; isa1, Delatte et al., 2005; lsf1, Comparot-Moss et al., 2010;
771
sex1, Yu et al., 2001; sex4, Kötting et al., 2009; pwd, Kötting et al., 2005). The lines 25
772
were as follows: amy3-2 (SAIL_613 D12); bam1-1 (SALK_039895); bam3-1 (CS92461);
773
bam4-1 (SALK_037355); gbss (GABI_914G01), isa1 (SALK_042704); lsf1-1
774
(SALK_100036); sex1-3 (Yu et al., 2001); sex4-3 (SALK_102567); pwd
775
(SALK_110814). Double mutants were selected from the F2 of crosses between esv1
776
and a second mutant line. Retention of both T-DNA insertions was checked for all
777
double mutant lines. Primers are listed in Supplemental Table 5.
778 779
Unless otherwise stated, Arabidopsis thaliana plants were grown on soil in growth
780
cabinets or controlled environment rooms with a 12 h light/12 h dark cycle, at 20°C and
781
at 150-180 µmol photons m-2 s-1 (metal halide lamps).
782 783
Generation and Mutagenesis of the Starvation Reporter Line
784 785
An A. thaliana starvation reporter line was established by transforming wild-type plants
786
with a construct consisting of a fusion of the starvation responsive promoter from
787
At1g10070 and the luciferase (LUC) gene (Graf et al., 2010). To create M0 seeds, 400
788
mg of homozygous T4 seeds of the reporter line were incubated in a 50-mL tube in 10
789
mL ethyl-methanesulphonate (EMS) solution [0.15% (v/v) EMS, 0.02% (v/v) Tween20]
790
on a rotating shaker at room temperature for 20 h. Seeds were washed with 10 mL
791
0.02% (v/v) Tween20 solution for 10 min on a rotating shaker. This washing step was
792
repeated 12 more times, and the seeds were then combined with 330 mL 0.1% (w/v)
793
aq. agar and pipetted on soil in 121 cm2 pots (10 mL per pot). The pots were held at 4°C
794
for 3 d and transferred to a controlled environment room. At 12 d old, five seedlings
795
were transferred to each of 1500 pots (121 cm2) in a greenhouse and allowed to set M2
796
seed. Seed from each pot was collected as a single pool.
797 798
Imaging of Bioluminescence
799 800
Seedlings or plants grown on soil were sprayed with luciferin solution [0.8 mM luciferin,
801
0.01% (v/v) Triton X-100] 24 h before imaging, then sprayed again 60 min before
802
imaging. Plants were transferred to the NightOwl CCD camera system (Berthold 26
803
Technologies; www.berthold.com) and bioluminescence was assayed using either
804
Indigo software (imaging settings: 1 min exposure time, 2x2 binning) or WinLight
805
software (imaging settings: 1 min exposure time, medium resolution, pixel binning 4x4,
806
single frame accumulation according to the manufacturer’s instructions).
807 808
Mapping of the ESV1 Gene
809 810
The esv1 mutant was out-crossed to the A. thaliana accession Landsberg erecta (Ler).
811
About 20 F1 plants were allowed to self-pollinate and set seed. F2 plants were grown for
812
10 d before identification of individuals showing bioluminescence by the end of the
813
normal night.
814 815
DNA was extracted from individual homozygous F2 plants. To obtain a rough map
816
position for the mutation, each plant was genotyped using genetic markers that were
817
distributed over the five chromosomes (Supplemental Table 1). Recombination
818
frequencies for each marker were calculated as the percentage of Ler polymorphisms
819
detected at that locus. To obtain a smaller interval, new polymorphic markers upstream
820
and downstream of the marker indicating the lowest recombination frequency were
821
analyzed. New markers were obtained using the A. thaliana mapping platform (Hou et
822
al., 2010; http://amp.genomics.org.cn/).
823 824
Identification of the ESV1 Gene
825 826
The mutation underlying the esv1 phenotype was identified by genome resequencing.
827
About 500 homozygous mutant plants were selected from the F2 of the cross between
828
the mutant and Ler. DNA was prepared from nuclei extracted from leaves. Library
829
construction from 5 µg of RNA-free gDNA, cluster generation, and sequencing on one
830
lane on the Illumina GAIIx platform were carried out by The Genome Analysis Centre
831
(Norwich, UK). The raw data were analysed as follows. Maq v0.71 (Li et al., 2008) was
832
used to align the 35.6M 100 base, paired-end reads against the TAIR8 Col-0 reference
833
sequence, producing about 28X coverage, and thus to generate a list of raw SNPs. The 27
834
maq.pl Perl script was employed to filter the SNPs on quality criteria, and the survivors
835
were used as input to a post-processing script that first eliminated SNPs that
836
corresponded to known Col-0/Ler polymorphisms (http://signal.salk.edu/atg1001/data/)
837
and then retained only EMS candidates from the remainder. The output of this script
838
was a GFF file that was loaded into a local instance of the GBrowse genome browser
839
(Stein et al., 2002), together with the TAIR8 pseudochromosome sequences and gene
840
model annotations, allowing visual inspection through a web browser. By interrogating
841
the GBrowse MySQL database with a Perl script using Bio::DB::GFF methods, a
842
genome-wide list of EMS candidates (G/C -> A/T) within annotated gene sequences
843
and inferred to induce either non-synonymous codon or donor/acceptor splice site
844
mutations was produced, which was then further refined based on chromosomal
845
location.
846 847
Quantification of Transcripts for Starvation Marker Genes
848
Quantitative reverse-transcription PCR (RT-qPCR) was used to measure transcript levels of
849
starvation marker genes, using the primers and methods described in Graf et al. (2010). Briefly,
850
total RNA was extracted from entire Arabidopsis rosettes harvested at the end of night, using an
851
RNeasy Plant RNA purification kit (www.qiagen.com). Following DNase treatment, 2 μg total
852
RNA was used for reverse transcription using RevertAid Reverse Transcriptase
853
(www.thermofisher.com), and qPCR analysis was performed using the Fast SYBR Green
854
master mix together with a 7500 Fast Real-Time PCR system (Applied Biosystems,
855
www.thermofisher.com). Transcript levels were calculated relative to the YLS8 housekeeping
856
gene. The primer pairs are listed in Supplemental Table 5.
857 858
Measurement of Starch and Sugar Contents
859 860
Starch and soluble sugars were extracted and quantified as previously described
861
(Critchley et al., 2001; Delatte et al., 2005; Martinis et al., 2014). Briefly, rosettes were
862
harvested into liquid nitrogen and ground to a fine powder in a ball mill. The powder was
863
suspended and agitated in ice-cold 0.7 M perchloric acid. Following centrifugation, the
864
pellet was washed three times in 80% (v/v) ethanol, resuspended in water, heated to
865
gelatinize the starch, digested with α-amylase and amyloglucosidase, and assayed 28
866
enzymatically for glucose. For sugars, the supernatant was neutralized, passed through
867
sequential cation- and anion-exchange columns (Dowex 50 and Dowex 1), and
868
analysed by High Performance Anion Exchange Chromatography with Pulsed
869
Amperometric Detection (HPAEC-PAD; www.dionex.com).
870 871
Visualization of Starch in Tissues
872 873
For iodine staining, tissues were decolorized in hot 80% (v/v) ethanol, rinsed in water,
874
stained in Lugol’s iodine solution and rinsed again. For visualization of starch in the
875
stem, tissue sections were stained using the modified Pseudo-Schiff Propidium Iodide
876
(mPS-PI) staining method described by Truernit et al. (2008). Stained sections were
877
imaged using confocal laser scanning microscopy, as described below.
878 879
Analysis of Starch Structure and Composition
880 881
The chain length distribution of amylopectin was profiled as described by Streb et al.
882
(2008). Briefly, starch in the pellet from the perchloric acid extraction (see above) was
883
debranched with isoamylase from Pseudomonas sp. (www.sigmaaldrich.com) and
884
pullulanase M1 from Klebsiella planticola (www.megazyme.com). The resulting glucan
885
chains were purified by passage through sequential cation- and anion-exchange
886
columns and analyzed by HPAEC-PAD.
887 888
Analysis of Granule Morphology, Amylose Content and Starch-Bound Phosphate
889 890
For the determination of amylose content, starch-bound phosphate, and granule
891
morphology by Scanning Electron Microscopy (SEM), starch granules were purified
892
from four-week–old Arabidopsis rosettes as described by Seung et al. (2015). The
893
apparent amylose content of the starch was determined using the iodine colorimetry-
894
based method described by Zeeman et al. (2002). Starch-bound phosphate was
895
quantified as described in Santelia et al. (2011). Hydrolyzed starch was
896
dephosphorylated with Antarctic phosphatase (New England Biolabs; www.neb.com), 29
897
and the phosphate released was quantified using malachite green. Granule morphology
898
was examined using a Merlin Field Emission Scanning Electron Microscope
899
(www.zeiss.com).
900 901
To visualize granule morphology within chloroplasts, segments from young leaves of
902
three-week-old plants were fixed in glutaraldehyde followed by osmium tetroxide and
903
embedded in Epon resin as described in Seung et al. (2015). Light microscopy images
904
of toluidine blue-stained sections were acquired on an AxioImager Z2 microscope fitted
905
with a 100× oil-immersion lens with 1.4 numerical aperture and an AxioCam
906
monochrome camera (www.zeiss.com).
907 908
For transmission electron microscopy, ultrathin (70 µm) sections were cut with a
909
diamond knife and placed on formvar carbon-coated copper grids, stained with 2% (w/v)
910
uranyl acetate and Reynold’s lead citrate and imaged with a FEI Morgagni 268 electron
911
microscope (www.fei.com). Pictures are representative of sections from two individual
912
plants per genotype.
913 914
Expression Vectors for YFP-Fusion Proteins in Planta and Plant Transformation
915 916
The coding sequences for Arabidopsis ESV1 and LESV were amplified from the full
917
length cDNA clones RAFL09-78-O20 and RAFL16-10-H06, respectively (RIKEN
918
Bioresource Centre; epd.brc.riken.jp), using primers flanked with attB recombination
919
sites. Primers are listed in Supplemental Table 5. The amplified inserts were then
920
recombined into the Gateway-compatible entry vector pDONR221 (Invitrogen;
921
www.thermofisher.com) and then recombined into the expression vector, pB7YWG2
922
(Karimi et al., 2002), downstream of the CaMV 35S promoter and in frame with the C-
923
terminal YFP tag. Expression constructs were transformed into Agrobacterium
924
tumefaciens strain GV3101.
925 926
Transient expression of YFP-tagged proteins in Nicotiana sylvestris (wild-type and pgm)
927
leaves was achieved by infiltrating A. tumefaciens cells into the abaxial epidermis 30
928
(Seung et al., 2015). Stable transformation of Arabidopsis was also carried out using the
929
floral-dipping method, as described by Zhang et al. (2006). Transformants were
930
identified in the T1 generation based on their resistance to the herbicide Basta.
931
Homozygous plants were identified in the T2 generation based on the segregation rates
932
of the Basta-resistance gene.
933 934
Detection of ESV1 and LESV Proteins by Silver Staining and Immunoblotting
935 936
For extraction of total (soluble and insoluble) proteins, two young leaves from individual
937
four-week-old rosettes were harvested and homogenized using a pestle in
938
microcentrifuge tubes in 300 µL extraction medium [40 mM Tris-HCl, pH 6.8, 5 mM
939
MgCl2, 2% (w/v) SDS, Complete Protease Inhibitor (www.roche.com)]. Insoluble debris
940
was pelleted at 20,000g. The protein concentration of the supernatant was determined
941
using the Pierce BCA Protein Assay kit (www.thermofisher.com), and the indicated
942
amounts of protein were loaded onto SDS-PAGE gels.
943 944
For the fractionation of soluble and insoluble proteins from N. sylvestris leaves, 7 mm
945
leaf discs were collected from transformed leaves at the end of the photoperiod three
946
days after infiltration, and snap frozen in liquid N2. Discs were homogenized using a
947
pestle in microcentrifuge tubes in 100 µL extraction medium [40 mM Tris-HCl, pH 6.8, 5
948
mM MgCl2, Complete Protease Inhibitor (www.roche.com)]. Insoluble debris was
949
pelleted at 20,000g. The pellet was washed once in extraction medium, then
950
resuspended in 100 µL SDS-PAGE loading medium [50 mM Tris-HCl, pH 6.8, 3% (w/v)
951
glycerol, 2% (w/v) SDS, 100 mM DTT and 0.005% (w/v) bromophenol blue) .The
952
suspension was heated at 95°C for 5 min, and insoluble debris were removed by
953
centrifugation. The supernatant was diluted with 10X SDS-PAGE loading medium.
954 955
Granule-bound proteins were extracted from purified starch granules (prepared as
956
described above) using the method described by Seung et al. (2015).
957
31
958
Silver staining was performed with the Silver Stain Plus kit (www.biorad.com). For
959
immunoblotting, proteins were transferred onto a PVDF membrane following SDS-
960
PAGE and probed with antisera specific to ESV1 or LESV. Antisera were raised in
961
rabbits against recombinant ESV1 or LESV proteins expressed in and purified from E.
962
coli. YFP-tagged ESV proteins were detected with an anti-GFP antiserum
963
(www.clontech.com). Plant actin was detected with a commercial monoclonal antibody
964
(Sigma A0480). Dilutions of antisera were as follows: Anti-ESV1, 1:1000; anti-LESV,
965
1:3000; anti-GFP, 1:5000; anti-actin,1:10000.
966 967
Confocal Laser Scanning Microscopy
968 969
Confocal laser scanning microscopy was carried out on an LSM 780 confocal
970
microscope (Carl Zeiss), with a 40X water-immersion lens (1.1 numerical aperture). For
971
the acquisition of YFP signal, the excitation beam was produced with an argon laser set
972
at 514 nm, and emitted light was captured between 518 to 557 nm. The
973
autofluorescence of chlorophyll was captured between 662 to 721 nm. Images were
974
processed with ImageJ software (http://rsbweb.nih.gov/ij/).
975 976
Phylogenetic Analysis
977 978
To build the phylogenetic tree, ESV1 and LESV sequences were retrieved from the
979
NCBI and 1000 plants (1KP; Johnson et al., 2012; http://www.onekp.com) databases
980
using BLASTp. The alignment was constructed using the MAFFT server (Katoh and
981
Standley, 2013) with the "Auto" alignment strategy. The tree was built using MEGA
982
software version 6 (Tamura et al., 2013), using an LG model, four gamma categories for
983
rate variation, a SPR level 5 method for heuristic search, and a neighbor-joining tree as
984
starting tree. 1,000 bootstrap replicates were used to assess branch support (branch
985
support
